Natural killer (NK) cells are lymphocytes of the innate immune system. They play a role in early host defense against tumors and infectious agents by direct cell killing mediated by their cytotoxic granules. In studies to characterize additional proteins associated with cytotoxicity, a novel protein whose expression is highly increased upon activation of NK cells was identified. This protein was named NK lytic-associated molecule (NKLAM). NKLAM is a granule transmembrane protein found in NK cells, cytotoxic T cells (CTL) and macrophages. Studies have shown a role for NKLAM in granule-mediated cytolysis. In macrophages, NKLAM is involved in the intracellular destruction of bacteria. To further assess the role of NKLAM, NKLAM deficient mice were generated. They have normal numbers of NK cells but exhibit 40-60% less NK activity in vitro than wild type mice. They also have higher numbers of lung metastases compared with wild type mice after injection with B16 melanoma cells. NKLAM is an E3 ubiquitin ligase, an enzyme involved in the process of protein ubiquitination. Upon degranulation or target cell stimulation, most NKLAM is lost from the NK cell. Our hypothesis is that upon activation of NK cells or CTL, NKLAM is released from the effector cell and enters the target cell. As a ubiquitin ligase, NKLAM ubiquitinates anti-apoptotic or growth associated molecules in the target cell, resulting in their degradation, thereby promoting target cell death. We have identified one protein that is a substrate for ubiquitination by NKLAM. The function of this novel protein, called uridine-cytidine kinase-like 1 (UCKL-1), is unknown;however, its homology to uridine kinases and overexpression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. RNA interference was used to downregulate UCKL-1 expression in K562 erythroleukemia cells. UCKL-1-depleted K562 cells proliferate more slowly, have increased cell death and enhanced susceptibility to apoptosis inducers and NK cell-mediated lysis compared with control treated cells. This proposal describes studies to: 1) Determine the role of NKLAM in cell-mediated cytotoxicity and in macrophage phagocytosis. Experiments are described to determine whether, upon degranulation, NKLAM enters target cells and enhances their death. 2) Characterize the role of UCKL-1 and other proteins that interact with NKLAM in cytotoxic function and tumor cell death. Studies are proposed to further elucidate the function of UCKL-1 in tumor cells. Since most E3 ligases have multiple substrates, studies are proposed to identify other NKLAM substrates. 3) Characterize NKLAM deficient mice to study the role of NKLAM in vivo. Studies are proposed to examine NKLAM deficient mice for NK, CTL activity and macrophage function. These mice provide the opportunity to evaluate a variety of tumor and pathogen models to determine where NKLAM plays a role. PUBLIC HEALTH RELEVANCE: NKLAM plays an important role in regulating and enhancing the anti-tumor and anti-microbial activities of natural killer (NK) cells, cytotoxic T cells (CTL) and macrophages. An understanding of these immunological processes is a critical first step in developing therapeutic interventions, and perhaps, ultimately, preventing cancer and infectious disease. These diseases are of great relevance to the health care system as the incidence of cancer is rising as the population is aging and new, resistant microbial pathogens are emerging and spreading.